The present invention relates to a new method of treating sepsis. The invention further provides possibilities for therapeutic and preventive treatment of Alzheimer""s disease.
Sepsis very generally comprises the clinical pictures which result from the presence of bacteria multiplying in blood. The direct cause of the symptoms are toxic substances which are released by the bacteria or released during lysis thereof. Gram-negative bacteria for instance produce lipopolysaccharides (LPS) as a component of their cell wall. These lipopolysaccharides are toxic in many circumstances. In principle they are bound to the cell and are only released when the cell lyses. Lipopolysaccharides are also referred to as endotoxins.
Infection with Gram-negative bacteria can result in a life-threatening disease which is initiated by specific binding of LPS to phagocytes, such as monocytes, macrophages and granulocytes (neutrophils). These are hereby activated and secrete various cytokines, such as tumor necrose factor-xcex1 (TNF-xcex1), interleukin 1 (IL-1), IL-6, IL-8, and other inflammation mediators. These compounds initiate a cascade of events, either directly or by activation of secondary mediators, which ultimately result in fever and disorders in the coagulation of the blood, vasodilation, organ failure and finally septic shock.
Different treatments for sepsis have already been proposed. Thus, for the treatment of specific Gram-negative sepsis, monoclonal antibodies are for instance used against endotoxin. Tests are currently also being carried out with recombinant BPI, a product of the neutrophils with a strong lipopolysaccharide-neutralizing effect.
Used for treatment of general sepsis are antibodies aimed against cytokines or antagonists for the soluble TNF (tumor necrose factor) receptor or for the interleukin-1 receptor.
Up to the present a totally satisfactory result has not yet been achieved with the known methods.
It is therefore the first object of the present invention to provide a new method of treatment and diagnosis of sepsis.
Surprisingly, it has been found that the per se known protein Serum Amyloid P component (SAP) is capable of binding to endotoxin. Binding of the circulating SAP to the phagocytes, and therewith activation thereof, is hereby prevented. In this manner SAP is capable of neutralizing the biological action of endotoxin.
The present invention therefore relates in a first aspect to the use of Serum Amyloid P component (SAP) for the preparation of a pharmaceutical composition for neutralizing lipopolysaccharide(s) in general and the treatment of sepsis in particular.
SAP is a member of the family of the pentraxins. Pentraxins are proteins with a characteristic pentameric organization of identical subunits which are ordered as single or double annular discs. Another member of this family is the C-reactive protein (CRP). CRP and SAP are both so-called xe2x80x9cacute-phase reactantsxe2x80x9d (APR), i.e. they are involved in the early phase of an inflammation process. Per species however, it is generally found that only one of the two acts as APR. For humans it is the case that the concentration of SAP in normal human plasma is approximately 30 xcexcg/ml. During inflammation reactions this level remains roughly the same, while the CRP level may well be increased a thousand-fold to 1 mg/ml, depending on the disease and the seriousness thereof.
It may be considered particularly surprising that a compound which plays no part as APR during inflammation reactions is in fact capable of neutralizing sepsis-causing endotoxin. It could be expected that the body itself would make use of this neutralizing capacity by increasing the plasma level of this compound in the case of infection with Gram-negative bacteria and the inflammation reactions resulting therefrom.
SAP is a relatively large protein. For particular ligands is known which of the amino acids of SAP are involved in binding, such as for instance for CRP, C4-binding protein (C4bp), Clq and Calcium ions. It has thus also been established for endotoxin that determined regions of the SAP molecule are involved in the binding. It may therefore be recommended to use only the specific binding parts of the protein for manufacture of a pharmaceutical preparation.
According to the invention it has now further been found that SAP fragments, which consist of at least a part of the amino acids 27-39 of SAP in the sequence occurring in SAP, are very successful in inhibiting the binding of LPS to monocytes.
According to a second aspect of the invention new peptides (SAP fragments) are therefore provided for use in neutralizing lipopolysaccharides, which peptides consist of a part of the amino acids 27-39 of SAP in the sequence occurring in SAP. Preferred peptides are the PEP 27-39, which consists of the amino acids 27-39, PEP 33-38 (amino acids 33-38), PEP 32-39 (amino acids 32-39), PEP 30-37 (amino acids 30-37) and PEP 29-36 (amino acids 29-36). Table 1 below shows the amino acid composition of the different peptides.
The advantage of SAP fragments is that they can be manufactured more simply owing to their smaller dimensions and can penetrate more easily into bodily tissues.
In the research which led to the present invention it was further inferred that LPS is possibly involved as environmental factor in the development of Alzheimer""s disease. Alzheimer""s disease coincides with particular forms of cancer, rheumatoid arthritis, diabetes and Down""s syndrome under the denominator xe2x80x98amyloidosisxe2x80x99. This is a collection of diseases which are characterized by extracellular deposits of normal or mutated proteins. The amyloid deposits in Alzheimer""s are ordered in a characteristic three-dimensional pattern of so-called xe2x80x9cbeta-pleated sheetsxe2x80x9d. The subunit protein component consists of the amyloid beta-protein (A beta-P). This is a small fragment of approximately 40 amino acids which is released by enzymes from the transmembrane beta-amyloid precursor protein (beta-APP). The processing of this precursor protein can take place in a number of ways and then results in a normally occurring soluble fragment or, under certain conditions via alternative proteases, an intact beta-fragment. The production of amyloid beta-protein is therefore in itself a normal physiological event and the existence of A beta-P can be demonstrated in the cerebral fluid (CFS) of healthy humans. However, the deposit of amyloid beta-protein is the primary event causing Alzheimer""s disease.
Other proteins are also associated with the amyloid deposits, including SAP and serum amyloid A (SAA). According to the invention it has now been found that both SAP and SAA can bind to LPS and are capable of neutralizing the biological activity of LPS. As such these two amyloid associated plasma proteins have no structural affinity.
According to the present invention it is now proposed that LPS enters into a binding with the serum amyloid proteins SAP and SAA, whereby the role of SAP and SAA in the initiation of amyloid deposits is influenced. It is suspected that through binding of LPS to SAP and SAA the occurrence of deposits is stimulated. The hypothesis now is that (chronic) bacterial infections, and particularly LPS as environmental factor, contribute to the development of Alzheimer""s disease. It is in fact suspected that the basis for Alzheimer""s is the alternative processing of the beta-amyloid precursor protein, which possibly takes place outside the brain in the circulation. There are indications that soluble amyloid beta-protein in plasma is associated with lipoproteins, in particular the VHDL and HDL3 fractions, in which it is complexed with the apolipo protein J (ApoJ). The ApoJ/amyloid beta-protein complex is capable of passing through the blood-brain barrier. In this manner the amyloid beta-protein enters the brain, where it is deposited. SAA is likewise an apolipo protein which is associated with the HDL fraction and particularly with the HDL3 subfraction.
It is further suspected that LPS also plays a part in the development of Alzheimer""s disease via an indirect route. Cytokines, such as interleukin 1 (IL-1) and interleukin 6 (IL-6), lead to over-expression of beta-amyloid precursor protein in the vessel wall and in microglia and astrocytes in the brain. This points to a role for the acute-phase response in the development of Alzheimer""s disease. LPS is a potent initiator of the acute-phase response and also initiates the production of IL-1 and IL-6. Because both cytokines result in more beta-amyloid precursor protein, an indirect role of LPS is assumed.
The SAP itself and fragments derived from SAP (peptides) with a strong LPS-binding and neutralizing action can therefore be of importance in eliminating the part played by LPS in the development of Alzheimer""s disease.
This influence can relate to the initiation as well as the further progression of the disease.
According to a third aspect the present invention therefore provides the use of SAP and/or SAP fragments thereof for the manufacture of a pharmaceutical composition for the therapeutic and preventive treatment of Alzheimer""s disease.
The pharmaceutical compositions, which according to the invention contain SAP and/or one or more SAP fragments (peptides) as active ingredient, will be particularly intended for parenteral, and then particularly intravenous use. The pharmaceutical compositions can be prepared by combining (i.e. mixing dissolving etc.) SAP and/or one or more SAP fragments with pharmaceutically acceptable excipients suitable for intravenous administration. The concentration of the active ingredient in a pharmaceutical composition can vary between 0.001% and 100%, depending on the nature of the treatment and the manner of administration. The dose of the active ingredient to be administered likewise depends on the administering route and application but can for instance vary between 0.01 xcexcg and 1 mg per kg body weight, preferably between 0.1 xcexcg and 100 xcexcg per kg body weight.
In addition to use in a pharmaceutical composition, SAP and/or the SAP fragments can also be used for diagnosis of infection with Gram-negative bacteria or sepsis. For this purpose the invention provides a diagnostic method for demonstrating the presence of endotoxin in blood or blood fractions, such as serum or plasma, comprising of bringing a carrier with SAP and/or endotoxin-binding SAP fragments bound thereto into contact with a blood sample for testing in order to enable binding of endotoxin to SAP(-fragments), removing non-bound material and visualizing and/or quantifying the binding between endotoxin and SAP(-fragments).
The invention further provides a diagnostic kit for performing the method, comprising a carrier having bound thereto SAP and/or endotoxin-binding fragments thereof, and means for visualizing and/or quantifying binding between endotoxin and SAP(-fragments).
The carrier can take different forms, such as a microtitre plate, a column, a membrane or beads. These latter can for instance be magnetic beads, such as Dyna-beads(trademark).
Binding of endotoxin to SAP or SAP fragments can be detected in different ways. Use can thus be made of a labelled antibody against endotoxin.
In the present application the terms xe2x80x98SAP fragment(s)xe2x80x99, xe2x80x98peptide(s)xe2x80x99 and xe2x80x98endotoxin-binding peptide(s)xe2x80x99 are used interchangeably.
The binding of SAP to endotoxin was found by xe2x80x9cfishingxe2x80x9d in plasma with magnetic beads having endotoxin bound thereto. The neutralizing action was tested on monocytes, neutrophils and in mice. In addition, SAP fragments were manufactured and the neutralizing action thereof was tested on monocytes. Details of these tests are described in the examples following hereinbelow, which are only given by way of illustration and are not intended to limit the invention in any way whatever.
Reference will be made in the examples to the following accompanying figures:
FIG. 1A shows an SDS-PAGE gel of LPS-Beads which are incubated with serum pre-incubated with free LPS.
lane 1: markers (respectively from top to bottom 92, 66, 45, 31 an d21 kDa),
lane 2: without pre-incubation with free LPS,
lane 3: pre-incubation with 100 xcexcg/ml free LPS
lane 4: pre-incubation with 10 xcexcg/ml free LPS
lane 5: pre-incubation with 1 xcexcg/ml free LPS.
FIG. 1B shows an SDS-PAGE gel of LPS-Beads incubation with serum (lane 1, negative control), commercially obtained SAP (lane 2) and markers (lane 3, molecular weight respectively from top to bottom 92, 66, 45, 31 and 21 kDa).
FIG. 2 shows the effect of SAP on LPS-binding to monocytes. Plotted on the X-axis is the concentration of SAP. The Y-axis shows the average fluorescence which represents the ReLPS-binding.
FIG. 3 shows the inhibition of the LPS priming of neutrophils by SAP in the presence and absence of LPS. In the graph the luminescence counts are plotted against the measurement time (in minutes).
FIG. 4 shows the effect of peptides (SAP fragments) according to the invention on the binding of LPS to monocytes. Plotted on the X-axis is the concentration of the peptides. The Y-axis shows the average fluorescence, which represents the ReLPS-binding.